Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer’s disease

Background Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer’s disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations. Methods Here, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-β (Aβ) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs. Results ENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aβ-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αβ toxicity and prevent synapse loss after Aβ treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aβ toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq. Conclusions Our work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer’s disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-024-03818-w.

MHz and were internally referenced to residual solvent peaks.Chemical shifts are reported in δ units, parts per million (ppm) and coupling constants (J) are given in Hz.

Chemical Synthesis
The synthesis of ENT-A011 and ENT-A012 is reported in Scheme 1 (Supplementary Data) and involves seven high-yielding steps starting from DHEA.Thus, Horner-Wadsworth-Emmons reaction of DHEA with triethylphosphonoacetate using sodium ethoxide (EtONa) as base gave the (E)-α,β-unsaturated ester 1 in 96% yield 26,27 , which was, in turn, reacted with tert-butyldimethylsilyl chloride (TBSCl) to afford the tert-butyldimethylsilyl-protected alcohol 2 in 93% yield.Selective reduction of the ester group in 2 using diisobutylaluminum hydride (DIBAL-H) gave the allylic alcohol 3 in quantitative yield, which was subjected to a Simmons-Smith cyclopropanation reaction in the presence of diiodomethane and diethylzinc to yield the (17S,20S)cyclopropyl derivative 4 in 60% yield after purification 28,29 .Compound 4 was subsequently oxidized with Dess-Martin periodinane (DMP) in dichloromethane (DCM) to afford the corresponding aldehyde 5 in 84% yield.Horner-Wadsworth-Emmons reaction of aldehyde 5 with diethyl(cyanomethyl)phosphonate in the presence of NaH afforded the α,β-unsaturated nitrile 6, as a mixture of E,Z geometrical isomers 6-E and 6-Z in 60:40 ratio, respectively 30 .Compounds 6-E and 6-Z were easily separated by flash column chromatography (FCC) and were deprotected separately using HF .Pyridine complex in dry CH2Cl2 to yield the final compounds ENT-A011 and ENT-A012, respectively, in quantitative yield.The geometry of the double bond in compounds ENT-A011 and ENT-A012 was confirmed by the corresponding coupling constants of the two vinylic protons of the unsaturated cyano functionality.In particular, in ENT-A011 they resonate at 5.31 ppm (d, J = 16.0Hz, 1H) and 6.30 ppm (dd, J = 16.0,10.2 Hz, 1H) and the coupling constant is equal to 16.0 Hz indicating the trans stereochemistry of the double bond (Supplementary Data, Figure S1).In addition, the cis stereochemistry in ENT-A012 is confirmed by the coupling constant (J) of the two vinylic protons being equal to 10.8 Hz [5.19 ppm (d, J = 10.8Hz, 1H) and 6.03 ppm (t, J = 10.8Hz, 1H)] (Supplementary Data, Figure S2).

Metabolic stability
Incubation conditions ensured linear metabolite formation with respect to reaction time and protein concentration (pooled human liver microsome concentration was set at 0.5mg/mL).To determine the oxidative (CYP-mediated) metabolic stability profile, 1mM NADPH served as a cofactor.ENT-A011 was tested at 1μM.Triplicate reactions took place at 37 °C in the presence of negative and positive controls (low vs. rapid clearance).Reactions were terminated after 60 minutes and readouts were recorded by Lionheart FX (BioTek) to determine the residual (%) of time zero (ENT-A011 depletion, Figure S7).In total, n=60 measurements per minute were acquired (t=60 minutes).

Isozyme-specific CYP450-metabolism
% Inhibition= (1-X/A) x 100% where X is the rate observed in the presence of test compound and A is the rate observed in the presence of negative (solvent, DMSO) control (Figure S7).

Computational Study of ENT-A011 TrkB Docking
Computational docking studies were performed to investigate the mechanism of action of the compound ENT-A011.The extracellular domain of TrkA has been previously reported to be a drug target [31][32][33] , while prior Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) experiments and molecular dynamics simulations have indicated that an analogue of ENT-A011, BNN27, binds at the interface of TrkA-D5 with NGF, thus bridging the heterodimer. 34Specifically, two binding sites, site 1a and site 1b, found at the interfaces of the two proteins were proposed to be the most probable binding sites for BNN27. 34Since there is high structural similarity between the TrkA and TrkB receptors, and ENT-A011 is an analogue of BNN27, the corresponding binding sites 1a and 1b of TrkB were identified in the present work and used for docking (Figure S6).The binding poses of the compound in the two sites show complementarity to the binding pockets.For site 1b, the docking pose has the 3β-hydroxyl group pointing towards the solvent region and the C17 steroid substituent facing towards the interface of TrkB-D5 and NT-4/5, thus providing a possible explanation for the selectivity of the substituent.The binding pose in site 1a is less solvent-exposed and preliminary molecular dynamics simulations showed that the compound bound in this pocket dissociates less readily than from site 1b (data not shown).Overall, the docking studies suggest two plausible interaction modes of ENT-A011 with TrkB that could lead to enhanced receptor activation.Biodegradation, metabolic, and toxicity liabilities can be identified early on and thus, inform structure-activity relationships (SAR).

ENT-A011 shows weak-to-moderate CYP inhibition
For this, after the administration of ENT-A011 at 1 μM, the activity of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (seven major human CYP450s) was assessed to determine a. the oxidative (CYP-mediated) metabolic stability profile in question and b. the enzyme metabolizing isoforms responsible (the test system consists of recombinant human CYP450 and CYP450 reductase; cytochrome b5 may also be present).
Herein, no concentration-dependent effects are anticipated, as our model depicts direct interactions between ENT-A011 and CYP450 isoenzymes.CYP450 substrates are known to change enzyme conformation, disrupt enzyme structure and/or function and/or block the enzyme active site, altering enzyme 35,36 .First, the catalytic activity of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was determined after the administration of ENT-A011 at 1 μM.Catalytic activity was assessed on the basis of the relative fluorescence of the enzymatic reaction product (Figure S6_A).ENT-A011 did not show strong effects on the enzyme (catalytic) activity of the seven major CYP450-isozymes tested herein.
Next, CYP450 (%) inhibition was determined.For the CYP450 isoenzymes tested, no product inhibition or mechanism-based inactivation was obtained.No solubility issues were reported for ENT-A011 (new chemical entities with poor solubility may result in artificially low CYP450 inhibition and thus, potential drug-drug interaction toxicities may escape our attention).CYP450 enzyme inhibition may result in unexpectedly high exposure of co-administered xenobiotics and hence, increase the risk for adverse effects.As depicted in Figure S6_B, ENT-A011 exhibits weak inhibition for CYP2D6, while it is a moderate inhibitor for CYP2B6, CYP2C9, and CYP2C19.For all CYP450 isoforms tested, we also assessed CYP450 (%) metabolic activity, expressed as residual % of time zero.Our findings are depicted in Figure S6_C, according to which ENT-A011 was overall metabolically stable (from t=0 to t=60 minutes).
Herein, we performed an isozyme-specific CYP450-study to delineate ENT-A011 interactions with each of the seven major CYP450s that account for xenobiotic biodegradation, metabolism, and toxicity as well as xenobiotic-xenobiotic and/or xenobiotic-host interactions.
ENT-A011 acts as a weak-to-moderate CYP450 enzyme inhibitor, yet showing no biodegradation, liver metabolism or safety issues.

ENT-A011 exhibits very slow depletion in human liver microsomes
The susceptibility of a test-compound to biotransformation is defined as metabolic stability.For the ranking of test compounds, several approaches can be followed including parent structure loss during metabolic reactions or their intrinsic clearance (CLint) and in vitro half-life (t1/2) values 37,38 .Herein, we chose the former.
ENT-A011 is very slowly depleted showing 93% residual of time zero at t=60 minutes.
Thus, ENT-A011 may correspond to low or medium intrinsic clearance classification bands.
For humans, a low intrinsic clearance classification band is defined by an CLint <8.6 μL/min/mg protein, whereas a high intrinsic clearance classification band is defined by an CLint >47.0 μL/min/mg protein.Low clearance test-compounds are characterized by enhanced exposure, prolonged half-life and reduced doses, predicted as suitable for once-daily dosing.

BDNF and ENT-A011 treatment effects on expression of target genes related to neuronal function
Various enriched shared terms show that both treatments lead to a positive regulation of neurotransmitters and their release.This includes "chemical synaptic transmission" and "signal release from synapse", with relevant target genes such as Bassoon (BSN), Complexin-2 (CPX-2)), Rab3-interacting molecule 2 (RIMS2) and Synaptotagmin-7 (SYT7) upregulated by both molecules, and additional related genes upregulated by BDNF such as Cholinergic receptor nicotinic alpha 4 (CHRNA4), Cholinergic receptor nicotinic beta 4 (CHRNB4) and Synapsin-1 (SYN1) or by ENT-A011 such as Glutamate decarboxylase 1 (GAD1), Glutamate receptor ionotropic AMPA 2 (GRIA2) and Metabotropic glutamate receptor 4 (GRM4).BSN is involved in the organization of the presynaptic active zone and regulation of neurotransmitter release 39 , CPLX2 plays a role in synaptic vesicle exocytosis and neurotransmitter release by modulating SNARE complex assembly 40 and RIMS2 regulates neurotransmitter release by interacting with Rab3 and modulating synaptic vesicle trafficking and docking 41 .What is more, CHRNA4 and CHRNB4 are structural components of acetylcholine receptors 42 , while GAD1, GRIA2 and GRM4 are involved in glutamate signalling [43][44][45] .
Additionally, some of these genes (e.g.functions in anchoring ion channels and transporters to the cytoskeleton 47 , KCND2 is a component of potassium channels 48 and CACNB3 is part of calcium channels 49 and ATP7A is involved in copper transport across membranes 50 .

Markers of iPSC-derived cortical neuronal progenitor identity
Immunostaining for selected markers (Sox2, Tbr2) to confirm differentiation quality is provided in Figure S13.Moreover, the expression of relevant markers is confirmed in the RNAseq data, including multipotency markers that are expected to be expressed in progenitors such as SOX2, PAX6, EOMES (Tbr2), FOXG1 and Nestin, as well as markers of neural differentiation such as MAP2, TUBB3 (Tuj1) and cortical markers TBR1, CUX1 and POU3F2 (Brn2).At the same time, iPSC markers POU5F1 (Oct4) and Nanog, or astrocytic marker GFAP are not expressed at detectable levels.
SYT7 and RIMS2) are specifically involved in calcium regulated neurotransmitter release ("calcium ion-regulated exocytosis of neurotransmitter" enriched for BDNF), with more examples of BDNF upregulated targets such as Heat shock 70kDa protein 5 (HSPA5) and Synaptotagmin-5 (SYT5) linked to "cellular response to calcium ion".From these examples, SYT7 functions as a calcium sensor for neurotransmitter release and regulates synaptic vesicle fusion with the plasma membrane46 .Glutamate receptor ionotropic NMDA 3A (GRIN3A) (along other targets mentioned) is an additional BDNF target that is also related to "regulation of neurotransmitter levels".ENT-A011 induced targets include more genes participating in "regulation of ion transmembrane transport / transporter activity", such as Ankyrin-3 (ANK3), Potassium voltage-gated channel subfamily D member 2 (KCND2), Copper-transporting ATPase 1 (ATP7A), Voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) and Reelin (RELN), with the latter also involved in "neurotransmitter-gated ion channel clustering", along with other interesting targets such as SLIT and NTRK-like family member 3 (SLITRK3) and Shisa homolog 7 (SHISA7).Interesting among these targets involved in ion channel regulation, ANK3

Figure S5 .Figure S6 :
Figure S5.ENT-A012 does not reduce cell death caused by serum deprivation in NIH-3T3 TrkB cells.Representative images (A) and quantification of Toxicity Assay (A') in NIH-3T3 TrkB cells after treatment with BDNF or compound ENT-A012 for 24h in serum free conditions.N=13, error bars represent S.E.M., Student's ttest against Control; **<0.01.The compound ENT-A012 do not induce TrkB and its downstream target Akt phosphorylation after 20 minutes treatment in NIH-3T3 TrkB stable expressed cells, representative blot (B) and quantification of TrkB and Akt Phosphorylation (B').Scalebar = 100μm

Figure S8 .
Figure S8.ENT-A011 does not activate the p75, TrkA and TrkC pathways.A. HEK293T cells were cotransfected with the plasmid cDNAs of p75NTR and TRAF6.Transfectants were exposed for 20 min to BDNF (500ng/ml) & the tested compound ENT-A011 (1μM) and lysates were immunoprecipitated with p75NTR-specific antibodies and then immunoblotted with antibodies against TRAF6.Total lysates were analysed for p75NTR, TRAF6 and actin expression by immunobloting.A'.Quantification shows that ENT-A011 does not activate p75NTR (one way ANOVA, no significance, Mean±SEM of triplicate measurements).B. HEK cells transfected with p75NTR were starved from serum and treated with ENT-A011 (1μM) or BDNF (500ng/ml) for 24hrs and subsequently subjected to CellTox assay.B'.Quantification shows that there is no significant difference between ENT-A011 treated group and negative control, Serum free.C. NIH-3T3 TrkC stable transfected cells were treated with Neurotrophin-3 (NT-3) or ENT-A011 for 20min and the lysates were immunoblotted with antibodies against pTrkC, TrkC, pAkt, Akt, pErk1/2, Erk1/2.Quantification of TrkC, Akt and Erk Phosphorylation (C') western blots.The compound ENT-A011 did not phosphorylate TrkC neither activate the downstream pathway D. PC12 cells were treated with NGF or ENT-A011 for 20 min, lysates were immunoblotted with pAkt, Akt, pErk1/2, Erk1/2.D'.Quantification of Akt and Erk Phosphorylation western blots.The TrkA signaling was not activated by the compound ENT-A011 in contrast to NGF.Scalebar = 100μm

Figure S11 .
Figure S11.Immunostaining of astrocytes cultures used in the current study against GFAP (red) and DAPI staining (blue).